User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/21: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 6: Line 6:
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==mm/dd/yy==
==04/21/15==
<!-- Precede finished items with a checkmark &#x2713; -->
<!-- Precede finished items with a checkmark &#x2713; -->
* Ryan - regulator assemblies
* Ryan - regulator assemblies
* Line item 2
* LCR Development - Phusion PCR




Line 33: Line 33:
# RpaR, 779 bp
# RpaR, 779 bp
# Receiver, Vector
# Receiver, Vector
Column clean-up
* tba


{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
{| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
|- valign="top"
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| bgcolor=#cfcfcf | Rxn 1-4
| rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
| bgcolor=#cfcfcf | Rxn 5
| rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
|-
|-
| DNA(plasmid) || 2.0 μL
| DNA || 10.0 μL
|-
|-
| 10X buffer || 1.5
| 10X buffer || 3.0
|-
|-
| EcoRI || 1.0
| EcoRI || 1.0
|-
|-
| PstI || 1.0
| XbaI || 1.0
|-
|-
| dH<sub>2</sub>O || 9.5
| dH<sub>2</sub>O || 15.0
|-
|-
| &nbsp; || 15 μL --> 37°C/ ~15 min.
| &nbsp; || 30 μL --> 37°C/ ~15 min.
|}
|}
Column clean-up
* tba


----
----

Revision as of 10:10, 21 April 2015

Karmella's BioBrick Cloning <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

04/21/15

  • Ryan - regulator assemblies
  • LCR Development - Phusion PCR


Ryan - Receiver plasmids, assembly

  • Stage 1 - insert Regulator ORFS
    • DONE - Phusion PCR-amplify ORFs (add 5'-E, 3'-X cut sites)
    • Insert ORF(E/X) into Vector (E/X). see Vector plasmid map
  • Stage 2 - insert Promoters
    • Cut & dephos promoters with E/X
    • Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling


Clean up PCR products

  • tba


Digests

  • E/X
  1. AubR, 851 bp
  2. BjaR, 776 bp
  3. BraR, 776 bp
  4. RpaR, 779 bp
  5. Receiver, Vector
Reagent Rxn 1-4 Rxn 5
DNA 10.0 μL
10X buffer 3.0
EcoRI 1.0
XbaI 1.0
dH2O 15.0
  30 μL --> 37°C/ ~15 min.


Column clean-up

  • tba




Assemblies

  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size


  • Digests (Fermentas FD)
    • Specific notes
Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


  • Dephosphorylation (Roche)
Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick #:1 (Pick #)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/04/21&oldid=881244"